Unnatural DNA used to encode unnatural proteins, all in otherwise normal cells.

To the best of our ability to tell, everything on Earth shares a few common features. It encodes information in DNA using four bases, A, T, C, and G. Sets of three consecutive bases are used to code for a single amino acid, and most organisms use a set of 20 amino acids to build proteins. These features appear everywhere, from plants and animals to bacteria and viruses, suggesting that they appeared in the last common ancestor of life on Earth.

This raises a question that comes up a lot in evolutionary studies: are these features used because they're in some way efficient, or did we end up stuck with them as a result of some historic accident?

A team of California-based researchers has been building an argument that it's an accident. And it's doing so by expanding life beyond the limitations inherited from its common ancestor. After having expanded the genetic alphabet to six letters, the team has now engineered a bacterial strain that uses the extra letters to put an unnatural amino acid into proteins.

New chemistry

The four bases in DNA are chemicals with a specific structure: flat rings with nitrogens and oxygens that can participate in hydrogen bonding. They form a specific number of bonds that allow them to pair: A forms two with T, while G forms three with C. These bonds hold together the double helix of DNA, but they also allow DNA to be transcribed into RNA, which uses a similar set of four bases (with T replaced by its close cousin U). And RNA uses this base pair to match sets of three bases to encode a specific amino acid.

Sets of three bases can encode 64 (4 x 4 x 4) possible items, but there are only 20 amino acids. So there's already room for some flexibility in terms of changing the genetic code. Unfortunately, all of life is already using all of those original 64 possible combinations to mean something else. So changing its meaning would require re-engineering the entire organism's genome.

Further Reading

Some researchers at Scripps, collaborating with a biotech company, decided to just expand the possibilities by adding two more bases that can interact with each other but not any of the four existing bases. To make sure that works, they got rid of the hydrogen bonding entirely; instead, the new bases interact through hydrophobic contacts—they stay stuck together because neither interacts well with the watery environment around them.

It's easy to draw out the bases' structures and see that they'd fit into DNA. And it's possible to show that they work in a test tube. But getting them to work in an organism is a different matter entirely.

Reimagining life

Cells don't make these artificial bases, and there are no enzymes that can. So the researchers have to supply them. But they're needed inside cells, which means they have to cross the membrane. It took a bit of searching for the authors to find a protein that would transport them across. Given all that, and the fact that some DNA with the artificial bases are already incorporated in them, bacterial cells would continue to use them.

Getting these new bases to be used as part of the genetic code is another, much more complicated matter. The three-base genetic code is translated using RNA, so the researchers needed to supply cells with an RNA version of the bases as well. The translation involves small transfer RNAs (tRNAs), which match the three-base code and are chemically linked to an amino acid. So a tRNA gene had to be supplied with one of the artificial bases. Finally, there needed to be an enzyme that chemically linked an amino acid to this tRNA.

To start with, the team decided to work with one of the amino acids that the cell uses already. This ensured that it already had the enzymes needed to link an amino acid to the tRNA—all they needed to do was add a tRNA gene with an artificial base. Once they did, the cell would take care of the rest.

To make sure this worked, the team also engineered a gene that encodes a fluorescent protein so that it also had an artificial amino acid in the middle of it. In normal cells that haven't been engineered, the cell has no way to deal with this, and the gene can't be translated into protein, so the cells won't glow. But add their engineered tRNA, and the cells could make the protein and would glow green. So, the system works.

But it works in the sense that it simply mimics what biology does already. Part of the point of this work is to get biology to do something new.

The researchers next took advantage of a rare species that uses a 21st amino acid (N6-[(2-propynyloxy)carbonyl]-l-lysine, since you asked). It has the enzymes to attach that amino acid to a tRNA. So the researchers took the gene for that enzyme and the gene for the tRNA, and they modified the tRNA to include an artificial base. When engineered into bacteria, this combination led them to insert this strange amino acid into the fluorescent protein, again allowing the cells to glow green.

The key thing here is that, collectively, this system takes life where it has never been before (at least since everything on Earth shared a common ancestor). The bases being used had never been in a living organism prior to this work. And they're being used to encode an amino acid that's only used by a handful of species in a completely separate domain of life. In doing so, the researchers have taken a genetic code that has 64 possible states and expanded it out to one that has 216.

Implications

There are a lot of implications to this work, so it's worth spending time to go through them. To begin with, it really does imply that life's choice of chemistry has been limited by a historic accident. Yes, everything about life requires nucleic and amino acids. But there's apparently a great deal of flexibility when it comes to what those nucleic and amino acids look like. That makes sense, given that the enzymes that make proteins already have to deal with 20 very distinct amino acids.

This also means we have a great deal of flexibility if we want to get organisms to use different amino acids. Enzymes can do many remarkable things given their relatively simple chemistry, but there's definitely the potential to expand on that—enzymes with phosphorus or boron in their structure, or metals chemically locked into a catalytic site. It opens up a whole world of chemistry, one that could see enzymes move into areas where they haven't seen a lot of use yet.

The lure of updating life's chemistry has been so strong that there's a group out there that's systematically editing a bacterial genome in order to free up a single three-base code for use with an artificial amino acid. The technique shown here is far more powerful, in that it opens up more than 150 new three-base codes. Which means we could potentially use multiple artificial amino acids in a single protein.

Which brings us to the technique's limitations. Since these artificial nucleic and amino acids aren't part of life's repertoire, no living organism knows how to make them. Which means they have to be supplied, and there has to be a way of importing them into a cell. For the artificial amino acids, it means there has to be a way to specifically link them to a tRNA for use, which means modifying an existing enzyme until it works with something new. That's not going to be easy.

But the key thing about getting this to work in living organisms is that it opens up an incredibly powerful tool for use: evolution. If we can make an organism's survival dependent upon our own poor implementation of some artificial chemistry, then a few hundred generations—meaning less than a week—will likely leave us with a finely tuned system.

This has me wondering: The size of the configuration space is no doubt proportional to the complexity required for an organism to survive and replicate. I'm going to throw a guess out there and say that the limitation on base count occurred wayyyy back before multicellular organisms, in which case the complexity of the environment was mostly driven by (and proportional to) raw thermodynamics and the overall chemical distribution (and thus ultimately, temperature and pressure). The only thing that bothers me is that it seems like it would be harder for a more complex system to survive in a hotter environment, so maybe the temperature-pressure relation isn't so simple...

opening paragraph is rather confusing. "dna is encoded using a set of 4 bases ... sets of THREE bases are used to codes for a single amino acid". What happened to the fourth base? Did you maybe mean "...sets of three base PAIRS"

Is it like early games where half a byte is used for an item identifier and the other half is used for the quantity of that item?

opening paragraph is rather confusing. "dna is encoded using a set of 4 bases ... sets of THREE bases are used to codes for a single amino acid". What happened to the fourth base? Did you maybe mean "...sets of three base PAIRS"

Is it like early games where half a byte is used for an item identifier and the other half is used for the quantity of that item?

It's confusing due to the nomenclature, yeah, but it means "there are four distinct bases, and a set of three 'slots,' each of which holds exactly one base (but it can be any of the four), encodes a single amino acid." But in practice, the word "base" is used for both.

>>To begin with, it really does imply that life's choice of chemistry has been limited by a historic accident.

Why? Just because they got something partially working in a hacked together way (which is awesome) doesn't mean that new implementation is more efficient than the old (at least nothing in the write up suggests they're anywhere close to being able to measure that).

There are 4 bases A G T C. Natural biology uses a set of 3 bases (for example CCT) to code for an amino acid (for example glycine). This is an example only and I doubt CCT is actually the code for glycine but I'm too lazy to look up the actual code. Hopefully this clarifies the situation.

I don't think it really matters, because the researchers have to supply the artificial base and the engineered tRNA for the artificial amino acid to be produced. If the bacteria escape, it won't be able to do anything the normal strain does, and the building blocks it needs are not found in the wild.

I don't think it really matters, because the researchers have to supply the artificial base and the engineered tRNA for the artificial amino acid to be produced. If the bacteria escape, it won't be able to do anything the normal strain does, and the building blocks it needs are not found in the wild.

Unless it evolves the necessary machinery to produce those bases by itself and goes on replicating, and it's the apocalypse!

I presented one of their previous papers for a journal club (these things never seem to go away, even after you're done training!). In the previous paper, they used transporters from algae to get the "exotic" nucleic acids into the cells as the bacteria replicate. They were able to get the bacteria to incorporate these new DNA bases into the genome and make them persist for subsequent generations.

If the article is TL;DR then here's my take. In this new paper, they figured out how to retrieve that information (see above) using new, custom-created tRNA's, and, as a proof of concept were able to get those tRNA's to ribosomes and successfully crank out some proteins (naturally occurring and non-natural). My guess is that they are currently searching for (or building it themselves!) transmembrane transporters to get "weird" amino acids into the cells. Once these are incorporated, they will have created organisms with an expanded genomic vocabulary, an expanded amino acid repertoire, a way to created "exotic" proteins, and finally, have it all be passed down from generation to generation.

Even more TL;DR: they're "almost" to the point of creating a entirely new species that uses more than just the ACGT in the DNA of everything else with the ability to use that expanded genome to encode for proteins using "weird" amino acids.

I don't think it really matters, because the researchers have to supply the artificial base and the engineered tRNA for the artificial amino acid to be produced. If the bacteria escape, it won't be able to do anything the normal strain does, and the building blocks it needs are not found in the wild.

Unless it evolves the necessary machinery to produce those bases by itself and goes on replicating, and it's the apocalypse!

Very cool. I'm amazed that just adding a gene for the new tRNAs worked. My (admittedly limited) understanding is that tRNA has other roles in the cell and can have rather arcane processes affecting it's expression. If they were able to just stick in the new gene and it got transcribed and worked, that's amazing. Did they look to see if there were other, unexpected side-effects of these new tRNAs?

"To make sure that works, they got rid of the hydrogen bonding entirely; instead, the new bases interact through hydrophobic contacts—they stay stuck together because neither interacts well with the watery environment around them."

Speaking from ignorance here: is the hydrophobic effect sufficiently stable to maintain a DNA strand over the long term?

"To make sure that works, they got rid of the hydrogen bonding entirely; instead, the new bases interact through hydrophobic contacts—they stay stuck together because neither interacts well with the watery environment around them."

Speaking from ignorance here: is the hydrophobic effect sufficiently stable to maintain a DNA strand over the long term?

Hydrophobic interactions are sufficiently stable to maintain cell membranes, so I wouldn't be surprised if they can maintain a DNA strand. Plus, it's not like they're replacing all of the bases with their novel base: there are still enough standard bases there to help out the bond.

opening paragraph is rather confusing. "dna is encoded using a set of 4 bases ... sets of THREE bases are used to codes for a single amino acid". What happened to the fourth base? Did you maybe mean "...sets of three base PAIRS"

Is it like early games where half a byte is used for an item identifier and the other half is used for the quantity of that item?

Previous replies were good, but what helps me understand is that there are 4 distinct bases which form the set of bases. These four bases appear in the DNA in sequences... a sequence/series of three bases codes for one of the 20 amino acids (or a stop, or probably a few other things I forgot). Adding the term "sequence" (noun, not the verb) in there may help.

The idea of an organism that can build metals sounds simultaneously awesome and incredibly scary.

There's a small sentence over there: "metals chemically locked into a catalytic site". Catalytic sites are the parts of the enzyme where the action takes place - where the enzyme does whatever it's meant to do. If you have "metal" bases in the genome and "metalic" active sites in the enzymes, you can have... Transformers. Living metal. The stuff Lemmy would write a song about where he alive

Jokes aside, it's an amazing piece of genetic engineering. What's more amazing is that it's all effectively a mapping expedition. They 're not currently building a real artificial organism. They map new territories, see what works and what doesn't. But once they get there... well the world will change very fast.

To further clarify my statement* and elucidate the "base pairs" portion. The DNA bases Adenine (A) pairs with Thymine (T) and Guanine (G) pairs with Cytosine (C). These pairs are important for error correction but NOT for coding. The pairs match up in a reciprocal fashion in the double helix. So the sequence..... ATTGCT.... would have a reciprocal on the other strand reading ...TAACGA..... But the code would be read by the molecular machinery as TAA (the machine would code for matching amino acid) the CGA would yield the amino acid that corresponds to that code and so on.

*There are 4 bases A G T C. Natural biology uses a set of 3 bases (for example CCT) to code for an amino acid (for example glycine). This is an example only and I doubt CCT is actually the code for glycine but I'm too lazy to look up the actual code. Hopefully this clarifies the situation.

>>To begin with, it really does imply that life's choice of chemistry has been limited by a historic accident.

Why? Just because they got something partially working in a hacked together way (which is awesome) doesn't mean that new implementation is more efficient than the old (at least nothing in the write up suggests they're anywhere close to being able to measure that).

I agree that the word "accident" is fraught with misunderstanding.

Life didn't "choose", it was literally inevitable, under the right circumstances. The laws of chemistry dictated that it would happen IF the environment contained the right base chemicals for it. Experiment after experiment proves that if you put oxygen, nitrogen, carbon and hydrogen together in the same place and throw in some energy (like static electricity, heat, something like that), you get biological stew - the proteins and components that can then combine with the same chemical properties that made them form in the first place into more complex biological complexes, until you start getting simple organisms like viruses.

Wash, rinse, repeat for a few billion years, and you get higher life forms like dinosaurs. MAN is the result of a natural disaster (the meteor the took out the dinosaurs allowed mammals to occupy new niches and evolve into new, larger varieties, which eventually created the hominid branch).

But there were no "accidents" involved with the beginning of life. It's just chemistry. If the environment was different, PERHAPS other kinds of life would evolve. That's the whole thing here, which I'm NOT seeing. The "take it back to the beginning" part and see if these things COULD HAVE HAPPENED NATURALLY, given a different environment.

What this looks like is someone trying to weld a truck bed from a Ford F150 onto the truck bed frame of a Toyota Tacoma. It may prove a point that such things are possible to exist, but without demonstrating/describing the environment in which such a thing could arise naturally, it's just not very likely to have happened in nature.

It's one thing to know that such things can exist. But much like genetically creating a unicorn, it's not going to ever be found "out there" in the real world (meaning anywhere in the universe) until we know what environmental conditions can bring one about. The differences between making something in the lab and finding it in the wild are usually pretty wide - especially with respect to biology.

>>To begin with, it really does imply that life's choice of chemistry has been limited by a historic accident.

Why? Just because they got something partially working in a hacked together way (which is awesome) doesn't mean that new implementation is more efficient than the old (at least nothing in the write up suggests they're anywhere close to being able to measure that).

I agree that the word "accident" is fraught with misunderstanding.

Life didn't "choose", it was literally inevitable, under the right circumstances. The laws of chemistry dictated that it would happen IF the environment contained the right base chemicals for it. Experiment after experiment proves that if you put oxygen, nitrogen, carbon and hydrogen together in the same place and throw in some energy (like static electricity, heat, something like that), you get biological stew - the proteins and components that can then combine with the same chemical properties that made them form in the first place into more complex biological complexes, until you start getting simple organisms like viruses.

Wash, rinse, repeat for a few billion years, and you get higher life forms like dinosaurs. MAN is the result of a natural disaster (the meteor the took out the dinosaurs allowed mammals to occupy new niches and evolve into new, larger varieties, which eventually created the hominid branch).

But there were no "accidents" involved with the beginning of life. It's just chemistry. If the environment was different, PERHAPS other kinds of life would evolve. That's the whole thing here, which I'm NOT seeing. The "take it back to the beginning" part and see if these things COULD HAVE HAPPENED NATURALLY, given a different environment.

What this looks like is someone trying to weld a truck bed from a Ford F150 onto the truck bed frame of a Toyota Tacoma. It may prove a point that such things are possible to exist, but without demonstrating/describing the environment in which such a thing could arise naturally, it's just not very likely to have happened in nature.

It's one thing to know that such things can exist. But much like genetically creating a unicorn, it's not going to ever be found "out there" in the real world (meaning anywhere in the universe) until we know what environmental conditions can bring one about. The differences between making something in the lab and finding it in the wild are usually pretty wide - especially with respect to biology.

Methionine (Met) is in green because it serves a special purpose (other than encoding methionine): all proteins start with AUG, that's how the machinery knows where to begin translation.

(Also, this table uses U instead of T because it's looking at the translation of mRNA into proteins, and RNA uses U instead of T. When transcribing from DNA to RNA, the Ts are all converted to Us, so there are only four options in either case.)

Hmmm, tinkering with the basic building blocks of life without really knowing how it works or what could happen, what could possibly go wrong? The movies The Stand, Life, or The Thing comes to mind.

There is no such thing as chance or accidents in a finite universe, just a very large number of variables, but there are way too many of them to guarantee that those working on this type of experiment won't have some sort of "accident" themselves if they encounter variables that they hadn't previously accounted for, which they no doubt will.

... That's the whole thing here, which I'm NOT seeing. The "take it back to the beginning" part and see if these things COULD HAVE HAPPENED NATURALLY, given a different environment.

What this looks like is someone trying to weld a truck bed from a Ford F150 onto the truck bed frame of a Toyota Tacoma. It may prove a point that such things are possible to exist, but without demonstrating/describing the environment in which such a thing could arise naturally, it's just not very likely to have happened in nature.

It's one thing to know that such things can exist. But much like genetically creating a unicorn, it's not going to ever be found "out there" in the real world (meaning anywhere in the universe) until we know what environmental conditions can bring one about. The differences between making something in the lab and finding it in the wild are usually pretty wide - especially with respect to biology.

Thing is, we don't know what the first organism and the respective environment really looked like. We only have clues, which are nothing to write home about when it comes to genetics. If we can't reconstruct it, we can't "take it back to the beginning" and craft a "new" evolutionary line out of it.

What we (they) can do is create unicorns and see how they survive and evolve (if at all). This is still valuable in studying Evolution, cause this time it is us who set "the beginning".

But again, this is merely a mapping expedition in unknown territory. And it's not alone. There are several teams and institutions around the world who work on similar subjects, although this is by far the most impressive piece of bioengineering I 've encountered. I wish I knew what DARPA's program on artificial life deals with.